Iqbal Zafar
Department of Clinical Laboratory Sciences, Medical Genetics and Hematology/Oncology, College of Applied Medical Sciences, King Saud Bin Abdulaziz University for Health Sciences, National Guard Health Affairs, Mail Code 3129, Riyadh 11426, Kingdom of Saudi Arabia ; Department of Hematology, Oncology and Pharmaco-genetic Engineering Sciences (HOPES) group, Health Sciences/Parasitology Research Laboratory, Department of Zoology, University of the Punjab, Lahore 54590, Pakistan ; Department of Biotechnology, Next-Generation Medical Biotechnology Division, School of Biological Sciences, University of Sargodha, Sargodha, Pakistan.
Indian J Hum Genet. 2014 Jan;20(1):64-8. doi: 10.4103/0971-6866.132758.
BCR-ABL fusion oncogene is a hallmark of Chronic Myeloid Leukemia (CML). It results due to translocation between chromosome 22 and chromosome 9 [t (9; 22)(q34; q11)]. It gives rise to translation of a 210 KDa chimeric protein (p210), leading to enhanced tyrosine kinase activity and activation of leukemogenic pathways, ultimately causing onset of CML. In case of CML, the classic fusions are b2a2 or b3a2, fusing exon 13 (b2) or exon 14 (b3) of BCR, respectively, to exon 2 (a2) of ABL. The type of BCR-ABL transcripts are thought to be have different prognosis and hence useful in clinical decision-making. The frequencies of different fusion oncogenes associated with leukemia can vary in different ethnic groups and geographical regions due to interplay of genetic variation in different ethnic populations, diverse environmental factors and living style. Moreover, earlier relevant studies from our region were carried out in small subset of patients. Therefore, objective of this study was to find out frequencies of different BCR-ABL splice variants in larger subset of CML patients.
A nested reverse transcriptase polymerase chain reaction (RT-PCR) was established to detect BCR-ABL splice variants in 130 CML patients. Sensitivity of RT-PCR and ability to detect BCR-ABL fusion gene in least possible time was studied.
BCR-ABL detection using our optimized RT-PCR protocol could be completed in 8 hours, starting from RNA extraction to Gel electrophoresis. Sensitivity of RT-PCR assay was of the order of 10(-6). Out of 130 Pakistani patients, 83 (63.84%) expressed b3a2 while 47 (36.15%) expressed b2a2 transcript.
Our RT-PCR was proved to be very quick to detect BCR-ABL fusion oncogene in CML patients within one working day. Because of its sensitivity, it can be used to monitor complete molecular response in CML. BCR-ABL RT-PCR and BCR-ABL splice variants frequency in our study differs from other ethnic groups. It shows that ethnic and geographical differences exist in BCR-ABL splice variant frequency, which may have a profound effect on disease biology as well as implications in prognosis and clinical management of BCR-ABL positive leukemias.
BCR-ABL融合癌基因是慢性髓性白血病（CML）的一个标志。它是由于22号染色体和9号染色体之间的易位[t(9;22)(q34;q11)]导致的。这会产生一种210千道尔顿嵌合蛋白（p210）的翻译，导致酪氨酸激酶活性增强以及白血病发生途径的激活，最终引发CML。在CML中，经典融合类型是b2a2或b3a2，分别将BCR的第13外显子（b2）或第14外显子（b3）与ABL的第2外显子（a2）融合。BCR-ABL转录本的类型被认为具有不同的预后，因此对临床决策有用。由于不同种族人群的基因变异、多样的环境因素和生活方式的相互作用，与白血病相关的不同融合癌基因的频率在不同种族群体和地理区域可能有所不同。此外，我们地区早期的相关研究是在一小部分患者中进行的。因此，本研究的目的是在更大的CML患者子集中找出不同BCR-ABL剪接变体的频率。
建立了巢式逆转录聚合酶链反应（RT-PCR）来检测130例CML患者中的BCR-ABL剪接变体。研究了RT-PCR的灵敏度以及在尽可能短的时间内检测BCR-ABL融合基因的能力。
使用我们优化的RT-PCR方案从RNA提取到凝胶电泳，8小时内即可完成BCR-ABL检测。RT-PCR检测的灵敏度约为10^(-6)。在130例巴基斯坦患者中，83例（63.84%）表达b3a2，47例（36.15%）表达b2a2转录本。
我们的RT-PCR被证明在一个工作日内就能快速检测CML患者中的BCR-ABL融合癌基因。由于其灵敏度，它可用于监测CML中的完全分子反应。我们研究中的BCR-ABL RT-PCR和BCR-ABL剪接变体频率与其他种族群体不同。这表明BCR-ABL剪接变体频率存在种族和地理差异，这可能对疾病生物学以及BCR-ABL阳性白血病的预后和临床管理产生深远影响。
